Biochemical and Mechanical Investigation of Cardiac Titin Isoforms

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Abstract

SUMMARY Background: Titin is a giant elastic protein of muscle sarcomeres. Titin molecules link the Z-disk with the M-line and have structural, elastic and signaling functions in myocytes. The primary structure determination of several titin isoforms and the mechanical characterization of different muscle tissues revealed that titin elasticity depends on the differential splicing of the titin spring region consisting of immunoglobulin-like domains, a so-called PEVK domain and larger unique sequence insertions like N2-B. The molecular weight of a titin isoform is correlated with its mechanical, spring-like properties: the smaller the isoform, the stiffer the spring. Heart muscles of mammalian organisms co-express two major classes of titin isoforms: the stiff N2B-titin and the compliant N2BA-titin. Sarcomeric stiffness is tuned by altering the expression ratio of N2BA:N2B titins, whereas the amount of total titin in a sarcomere likely is constant owing to stoichiometric constraints. Objectives of the study: 1) To determine the patterns of titin isoform expression in different muscle tissues using MDa-range high-resolution gel electrophoresis and Western blotting; 2) To understand the functional significance of the expression of various cardiac titin isoforms; 3) To look for variations in cardiac titin expression in diseased myocardium; 4) To establish conditions/factors determining different expression patterns of cardiac titin; 5) To understand consequences of pathological changes in titin protein expression for the heart. Methods and Results: N2BA to N2B titin isoform ratio was determined by loose-gel electrophoresis. The titin isoform ratio differed between: 1) Hearts from different mammalian species; 2) Various regions of the same heart; 3) Diseased and normal human hearts. Western blotting using sequence assigned anti-titin antibodies confirmed the identity of the titin bands. The N2BA proportion varied from ~5% in rat left ventricle to almost 70% in cow right ventricle. The N2BA:N2B ratio was generally higher in the right ventricle than in the corresponding plane of the left ventricle and decreased from the base to the apex of a given heart (assessed in goat and rabbit). Titin isoform expression was altered under disease conditions: human heart transplants due to coronary artery disease (CAD) exhibited an average N2BA:N2B ratio of 47:53, whereas normal donor hearts had a ratio of ~30:70. Increased expression of larger N2BA titin isoforms was also seen in failing myocardium of dilated cardiomyopathy (DCM) patients. Coexpression of N2BA-titin and N2B-titin in a sarcomere was demonstrated by immunofluorescence microscopy. A regular cross-striated staining pattern for titin on tissue sections of CAD-transplant hearts indicated uniform changes of titin expression instead of titin structural damage. The functional relevance of the observed changes in titin isoform expression was estimated in mechanical experiments on isolated myofibrils from human hearts. Diseased (CAD, DCM) human myofibrils expressing elevated N2BA proportions had lowered passive stiffness compared to non-failing human myofibrils. Thus, sarcomeres can modify their passive tension by adjusting the N2BA:N2B titin expression ratio. Failing human hearts, even if they are globally stiffened (collagen upregulated), have more compliant myofibrils than normal donor human hearts. Titin isoform switching was also studied in a rat model of myocardial infarction (ligature of left anterior descending coronary artery). Titin gels showed that 43% of diseased hearts displayed a distinct N2BA-titin band, compared to only 14% of the hearts of sham-operated control rats, suggesting an isoform switch had occurred in this heart failure model. Conclusions: An improved titin detection method by modified 2% SDS-polyacrylamide gel electrophoresis revealed the presence of multiple titin isoforms in different tissues. Results established that the elastic diversity of titin is altered in human heart disease and during development. The shift towards expression of more compliant titin isoforms in human heart failure alters mechanical properties of the cardiomyocytes, in particular the passive stiffness. The disease-induced shift in titin isoform ratio may also impair active contraction, e.g. by interfering with the ability of the heart to use the Frank-Starling mechanism.